Abstract
Two-photon excited fluorescence (TPEF) microscopy and fluorescence lifetime imaging (FLIM) are powerful imaging techniques in bio-molecular science. The need for elaborate light sources for TPEF and speed limitations for FLIM, however, hinder an even wider application. We present a way to overcome this limitations by combining a robust and inexpensive fiber laser for nonlinear excitation with a fast analog digitization method for rapid FLIM imaging. The applied sub nanosecond pulsed laser source is synchronized to a high analog bandwidth signal detection for single shot TPEF- and single shot FLIM imaging. The actively modulated pulses at 1064nm from the fiber laser are adjustable from 50ps to 5ns with kW of peak power. At a typically applied pulse lengths and repetition rates, the duty cycle is comparable to typically used femtosecond pulses and thus the peak power is also comparable at same cw-power. Hence, both types of excitation should yield the same number of fluorescence photons per time on average when used for TPEF imaging. However, in the 100ps configuration, a thousand times more fluorescence photons are generated per pulse. In this paper, we now show that the higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to not only use a single pulse per pixel for TPEF imaging but also to resolve the exponential time decay for FLIM. To evaluate the performance of our system, we acquired FLIM images of a Convallaria sample with pixel rates of 1 MHz where the lifetime information is directly measured with a fast real time digitizer. With the presented results, we show that longer pulses in the many-10ps to nanosecond regime can be readily applied for TPEF imaging and enable new imaging modalities like single pulse FLIM.
Highlights
Two-photon excited fluorescence (TPEF) microscopy is a powerful technique in bio-molecular imaging to gain a greater understanding of processes ongoing on a cellular level[1]
We show that the higher number of fluorescence photons per pulse combined with a high analog bandwidth detection makes it possible to use a single pulse per pixel for TPEF imaging and to resolve the exponential time decay for fluorescence lifetime imaging (FLIM)
We show that longer pulses in the many-10ps to nanosecond regime can be readily applied for TPEF imaging and enable new imaging modalities like single pulse FLIM
Summary
Two-photon excited fluorescence (TPEF) microscopy is a powerful technique in bio-molecular imaging to gain a greater understanding of processes ongoing on a cellular level[1]. Have the different fluorophores different lifetimes but more importantly, the lifetime changes with its neighboring molecules and can give information on the molecular bounds With this wealth of information, TPEF microscopy combined with fluorescence lifetime imaging (FLIM) is a valuable tool for researchers in bio-molecular science [3,4,5]. The work horses for this kind of excitation are currently ultra-short pulse Ti:Sa lasers with pulse durations in the order of ~100 fs and a repetition rate of ~100 MHz [6] As these lasers tend to be bulky, expensive, and not fiber compatible, TPEF imaging is usually done only in sophisticated optics labs. For rapid assessment of large areas or for monitoring dynamic processes, this is often too slow
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